The present invention relates to a high affinity polypeptide having a binding activity to inositol 1,4,5-trisphosphate, to a gene encoding the polypeptide, to a recombinant vector including the gene, to a transformant including the vector and to a method for producing the high affinity polypeptide having a binding activity to inositol 1,4,5-trisphosphate.
Inositol 1,4,5-trisphosphate (hereinafter, also referred to as xe2x80x9cIP3xe2x80x9d) is one of second messengers which are produced by inositol phospholipid metabolism activated in response to an extracellular stimului such as hormones, growth factors, neurotransmitters or the like. IP3 is a substance that induces the increase of intracellular calcium concentration. The IP3-induced calcium increase is a crucial and highly universal signal transmission mechanism that is involved in many cell functions in a wide variety of animals. For example, IP3 controls many physiological functions such as fertilization, blastogenesis, development and differentiation, cell growth, secretion, immune system, muscle contraction, and cranial nerve functions (gustation, vision, memory, learning, etc.) in diverse organisms, for example, invertebrate such as nematoda (nemathelminthes), Drosophila (arthropoda) and cuttlefish (mollusca), and vertebrata such as mouse and human.
On the molecular level, this mechanism is initiated by the binding between an IP3 and its target, an IP3 receptor. Specifically, when the IP3 binds to the IP3 receptor (a calcium channel susceptible to IP3) present in an intracellular calcium-storing site (endoplasmic reticulum, etc.), the channel opens and releases calcium from the calcium-storing site into the cytoplasm, thereby controlling the activities of calcium-dependent proteins and enzymes.
Heparin, adenophostin (a kind of fungal metabolite) and Xestospongin (a kind of sponge metablite) are examples of substances that might affect the signal transmission by the IP3-induced calcium. However, although heparin inhibits the binding between the IP3 and the IP3 receptor, its specificity is low since there are various targets in the cell. Adenophostin is an antagonistic agonist of the binding between the IP3 and the IP3 receptor, and is a powerful activator of the IP3 receptor channel. However, its use is limited since its yield from fungus is low and it cannot transport across the membrane. Xestospongin has recently been reported as an inhibitor of the IP3 receptor channel that does not influence the binding of IP3. Again, its yield is low and there are still questions remaining as to its specificity. Thus, currently, there is almost no substance that is considered to effectively act on IP3-induced calcium signal transmission. In particular, there has been no substance or system that inhibits IP3-induced calcium signal transmission by specifically trapping IP3 that has increased on the cell level.
The present invention provides a high affinity polypeptide having a binding activity to inositol 1,4,5-trisphosphate, a gene encoding the polypeptide, a recombinant vector containing the gene, a transformant containing the vector and a method for producing the high affinity polypeptide having a binding activity to inositol 1,4,5-trisphosphate.
In order to solve the above-described problem, the present inventors have gone through intensive studies and have succeeded in isolating a high affinity polypeptide having an extremely high binding activity to IP3 from a protein including a part of the N-terminal amino acid region of an IP3 receptor.
The present invention provides a recombinant polypeptide of the following (a), (b) or (c):
(a) a polypeptide comprising an amino acid sequence shown in SEQ ID NO: 2;
(b) a polypeptide comprising an amino acid sequence having deletion, substitution or addition of at least one amino acid in the amino acid sequence shown in SEQ ID NO: 2, and having a high affinity binding activity to inositol 1,4,5-trisphosphate; or
(c) a polypeptide having at least 70% homology with the amino acid sequence shown in SEQ ID NO: 2, and having a high affinity binding activity with inositol 1,4,5-trisphosphate.
The present invention also provides a gene coding for a polypeptide of the above (a), (b) or (c); or a gene coding for a polypeptide having at least 70% homology with the gene and having a high affinity binding activity with inositol 1,4,5-trisphosphate.
The present invention further provides a gene comprising DNA of the following (d) or (e):
(d) DNA of a nucleotide sequence shown in SEQ ID NO: 1; or
(e) DNA of a nucleotide sequence having at least 70% homology with the DNA of the nucleotide sequence shown in SEQ ID NO: 1, and coding for a polypeptide having a high affinity binding activity with inositol 1,4,5-trisphosphate.
The present invention provides a recombinant vector comprising any one of the above-described genes.
The present invention also provides a transformant comprising the above recombinant vector.
The present invention further provides a method for producing any one of the above-mentioned polypeptides, the method comprising: culturing the above-mentioned transformant; and collecting, from the obtained culture, a polypeptide having a high affinity binding activity to inositol 1,4,5-trisphosphate.
This and other advantages of the present invention will become apparent to those skilled in the art upon reading and understanding the following detailed description with reference to the accompanying figures.
This specification includes part or all of the contents as disclosed in the specification and/or drawings of Japanese Patent Application No. 10-242207 which is a priority document of the present invention.